The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
In the past, few decades' tuberculosis has emerged as a cause of considerable human mortality worldwide. It has been found that there is a steady increase in the frequency of mycobacterial strains, which have developed resistance against one or more anti-mycobacterial agents commonly used in treatment. Therefore, to overcome the situation there is a need to have better drug intervention strategies, which can be achieved by identification of new drug targets. In this consequence, the enzyme peptide deformylase is involved in deformylation of nascent polypeptides, which appears to be a mandatory step in mycobacterial protein synthesis in general. Therefore, any biotic/abiotic factor(s) inhibiting this enzyme may prevent protein synthesis in general in mycobacteria and thus specifically inhibits its growth.
Drug resistance in pathogenic microorganisms has emerged as a great threat to public health worldwide. Although there is large number of antibiotics used, the variety of target they inhibit is very limited. Consequences of the prolonged and excessive use of these antibiotics outlay multi-drug resistance in the pathogenic microorganisms. Therefore, in order to diversify the spectrum of antimicrobial agents, there is an urgent need to frame new intervention strategies, based on rational approaches, which would allow improved drug design.
Protein synthesis has always been proven to be a rich source of targets for antimicrobials. In contrast to the eukaryotes, protein synthesis in prokaryotes is initiated with N-formyl-methionyl-tRNA leading to formylation of all nascent polypeptides at the amino-terminal end. The N-formylmethionine, however, is not retained in mature proteins of eubacteria and has been reported to be deformylated by peptide deformylase. This formylation/deformylation event appears to be a mandatory step in eubacterial protein synthesis and therefore, the importance of this enzyme has long been envisaged.
Available genome sequencing data revealed the presence of putative gene encoding the peptide deformylase (def) throughout the eubacterial lineage including pathogens like Mycobacterium tuberculosis (NCBI general identification GI: 38490165; SEQ ID NO: 8), Staphylococcus aureus, (NCBI general identification GI: 57651784 SEQ ID NO: 1) Streptococcus pneumoniae (NCBI general identification GI: 16272565 SEQ ID NO: 2), Haemophillus influenzae (NCBI general identification GI: 16272565; SEQ ID NO: 3), Leptospira interrogans (NCBI general identification GI: 14626937; SEQ ID NO: 4), Enterococcus feacelis (NCBI general identification GI: 29377524; SEQ ID NO: 5), Helicobacter pyroli (NCBI general identification GI: 49089809; SEQ ID NO: 6) and Bacillus subtilis (NCBI general identification GI: 16078635; SEQ ID NO: 7). etc. Earlier studies have shown the identification and use of various compounds or preparations and their derivative inhibiting the activity of peptide deformylase in various microorganisms (Patent no: WO0138561, WO2005026133, WO2005037272, WO2005092872 etc).
The article by Tomioka, H (Prospects for development of new antituberculous drugs. Kekkaku. August; 77[8] 573-84, 2002) in general describes the pharmacological status of certain new derivatives of existing drugs such as rifamycin (rifabutin, rifapentine, and rifalazil), fluoroquinolones (ciprofloxacin, ofloxacin, sparfloxacin, levofloxacin, gatifloxacin, sitafloxacin, moxifloxacin, and others), and new macrolides (clarithromycin, azithromycin, and roxithromycin). This review also discusses the importance of the development of new antimycobacterial, especially antituberculous agents including oxazolidinone (PNU-100480), 5′-nitroimidazole (CGI 17341), 2-pyridone (ABT-255), new riminophenazines, nitroimidazopyran (PA-824), new ketolides (ABT-773, telithromycin) and defensins (human neutrophil peptide-I). Moreover, authors have described the possibility of designing inhibitors (certainly one of the strategy could be an antisense technology) specific to mycobacterial genes encoding certain metabolic enzymes or virulence factors as a new drug targets. In fact, use of antisense oligonucleotides to shut down the expression of mycobacterial genes is a very familiar technique (For reference: Harth et al., Proc. Natl. Acad. Sci. U.S.A. 99, 15614-15619, 2002).
The present invention highlights the importance of Insertion sequence specifically present in mycobacterial peptide deformylase (consisting of amino acids 74-85, (Please refer FIG. 1 and FIG. 2) responsible for maintaining the functionality of the enzyme (FIG. 5, where it is shown that deletion mutant of this region did not show any enzyme activity). Furthermore, the use of antisense oligonucleotide (complementary to the corresponding nucleic acid of SEQ ID NO: 21) against the insertion region reduces the expression of peptide deformylase enzyme (as shown in FIG. 8 by western blotting using anti-mPDF antibody), which in turn leads to the growth inhibition of mycobacteria in culture (FIG. 6A and left panel of FIG. 7). These results therefore describe the novelty of the insertion region of mycobacterial enzyme, which we have invented, in terms of the possibility of designing inhibitors based on this insertion region (Antisense molecule has been used to elucidate the importance of the region in contributing mycobacterial growth).
In another article by Cynamon, et al. 2004. Journal of Antimicrobial Chemotherapy. 53: 403-405 it is recited that actinonin an antibiotic isolated from class Actinomycetes as well as BB3497 (a hydroxamic acid derivative of actinonin) showed inhibition for PDF enzyme activity from different microorganisms by binding to the active site. The mentioned article describes the inhibitory effect of BB3497 on the growth of mycobacteria in culture possibly by inhibiting PDF enzyme activity. Cynamon, et al. 2004 in their paper showed a known peptide deformylase inhibitor inhibits mycobacterial growth. On the other hand, we initiated our studies through characterization of mPDF and established that despite the commonality, it is distinctly different from other bacterial homologues. Sequence analysis of peptide deformylase of M. tuberculosis revealed the presence of characteristic insertions (residues 74-85) between motifs I and II (FIG. 1). The result of the instant application with deletion mutant indicates the contribution of this region towards functionality of the enzyme (FIG. 5). Among PDFs characterized to-date, our analysis revealed that the constituent amino acids of the insertion region is typical of mycobacterial species (FIG. 2). Moreover, using 5′-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against this insertion region, we showed inhibition of mycobacterial growth in cultures, establishing the importance of this region (FIG. 6A). Furthermore, antisense oligonucleotide directed against insertion sequence specific to mycobacteria has no effect on the functionality of PDF enzyme from other bacteria such as Escherichia coli (as shown in right panel of FIG. 7). Thus our results clearly establish that the antisense oligonucleotide directed against the insertion region specifically inhibits the expression of the mycobacterial peptide deformylase enzyme (FIG. 8) and therefore, the growth of the mycobacteria (FIG. 6A and left panel of FIG. 7). Hence, we claim that we have identified a region in mycobacterial peptide deformylase enzyme (amino acid residues 74-85), which is important towards the functionality of the enzyme in mycobacteria. Any molecule (biotic or abiotic) that interacts with this region of the mycobacterial enzyme and affects the expression or production of this enzyme can inhibit mycobacterial growth. (We established this by using an antisense oligonucleotide directed against this region. So it is an approach to validate our conclusion/invention). Therefore, this region (amino acid residues 74-85), which we have identified in mycobacteria for the first time as well as established its importance (FIGS. 5 to 8) is definitely a drug target for development of antimycobacterials.
Huntington, K. M. 2000. Biochemistry. April 18; 39[15]: 4543-51 reports the recent information on the whole genome of various pathogenic bacteria including M. tuberculosis certainly provides a good platform to promote the progression in the identification of genes that code for new drug targets. Essential genes encoding proteins involved in metabolism and survival of pathogenic microorganisms are always being preferential vaccine candidates. Similarly, peptide deformylase is among one of the essential enzyme, which is involved in posttranslational modification of N-formylated polypeptides in prokaryotes (Mazel et al., 1994, Margolis et al., 2000 and 2001). It has been characterized as either zinc or ferrous containing metalloprotease in many eubacteria. Its essential character in bacterial cells makes it an attractive target for antibacterial drug design. Authors in the above mentioned article showed that they have rationally designed and synthesized a series of peptide thiols that act as potent, reversible inhibitors of purified recombinant peptide deformylase from Escherichia coli and Bacillus subtilis by binding to the active site. The PDF inhibitors induce bacterial cell lysis and have been tested to be bactericidal to B. subtilis, Staphylococcus epidermidis, Enterococcus faecalis, and E. coli. However, the present invention is specifically focused to M. tuberculosis. Authors have nowhere mentioned the effect of these compounds on the activity of purified mycobacterial enzyme as well as on the growth of mycobacteria. On the other hand, our work specifically deals with mycobacterial PDF and claims for the first time that an insertion sequence specific to mycobacterial enzyme could be focused to develop new antimycobacterials.
Recently, we have PCR amplified the 594 base pair def gene from M. tuberculosis and following cloning in pET28c vector, expressed it as a histidine-tagged fusion protein in Escherichia coli (Saxena and Chakraborti, Biochem Biophys Res Commun (332): 418-425, (2005)). Although atomic absorption spectroscopy revealed that mPDF was a Fe+2-containing enzyme, its activity was very stable at 30° C. with a half-life of ˜4 h. Furthermore, it maintained its distinction by exhibiting resistance to oxidizing agents, like H2O2 (Saxena and Chakraborti, Biochem Biophys Res Commun 332: 418-425, 2005); Saxena and Chakraborti, J. Bacteriol 187: 8216-8220 2005). Since conversion of Fe+2 to Fe+3 by environmental oxygen resulted in inactivation of this metallo-protease in E. coli (Rajagopalan, et. al., J. Biol. Chem. 36: 13910-13918, 1997), this seems to be an important observation considering the fact that M. tuberculosis has to cope up with oxidative stress for its survival within the host as a successful pathogen.
This led us to characterize the mycobacterial peptide deformylase enzyme. In contrast to other studies (Patent no. WO02074903), our invention is related to use of an antisense oligonucleotide complementary to specific nucleotide region of the mycobacterial peptide deformylase gene (def), which inhibits enzyme activity, as well as the growth of this microorganism in culture establishing its essentiality and its potential as a drug target.